BIOLOGICAL ACTIVITY OF MEDROGESTONE A NEW ORALLY ACTIVE PROGESTIN C. REVESZ and C. I. CHAPPEL Ayerst Research Laboratories, Montreal, Canada [Received 12th February 1966) Summary. A new synthetic orally active progestational agent 'Medro- gestone' was investigated and its activity compared to progesterone and medroxyprogesterone acetate (map). Medrogestone fulfils the criteria of a pure gestagen, it has a spectrum of biological activity similar to that of progesterone but unlike progesterone, it is orally active. Medro- gestone is free of undesirable side effects in experimental animals. It is being investigated clinically as an oral progestin (Carter, Faucher & Greenblatt, 1964). INTRODUCTION The lack of oral effectiveness of progesterone inspired the search during recent years for progestational compounds which would be orally active. A number of such compounds have been synthesized, tested in animals and are now widely used in menstrual irregularities, habitual, threatened abortion, cycle regulation, treatment of infertility and/or in combination with oestrogens as contraceptives. However, many of these substances in addition to their pro¬ gestational activity produce certain side-effects such as androgenicity, mascu- linization of female foetuses and adrenal atrophy. The desirability of an orally active progestin which would be free from these objectionable properties led to the synthesis of a series of derivatives of 17-methylprogesterone (Deghenghi & Gaudry, 1961; Deghenghi, Revesz & Gaudry, 1963) and their testing as progestational agents. The biological activity of 6-methyl-6-dehydro-17- methylprogesterone, which appeared to be the most promising member of this series, forms the subject of this paper. It has the following structural formula: CH, * Generic name of 6-methyl-6-dehydro-17-methylprogesterone. Trade name in U.S.A. is Colprone, also known as -62022. t Present address: Bio-Research Laboratories, Pointe-Claire, Quebec, Canada. 473 Downloaded from Bioscientifica.com at 10/02/2021 12:28:16PM via free access 474 C. Revesz and C. I. Chappel METHODS Progestational activity McPhail's (1934) modification of the Clauberg test was employed using progesterone and medroxyprogesterone acetate (map) as reference compounds. Immature female rabbits were primed with a total dose of 15 ^g oestrone given over a period of 6 days. From the 7th to the 12th day of the experiment the test compound was given daily, subcutaneously (in 0-2 ml of sesame oil) or orally (in 2 ml of sesame oil). Twenty-four hours after the last dose was given the animals were killed and sections removed from the proximal, middle and distal portions of each uterine horn. Specimens were fixed in Bouin's solution, sectioned at 5 µ and stained with Harris's haematoxylin and eosin stain. The sections were evaluated according to McPhail's grading system. Duration of action ofprogestational activity The duration of effect of a single dose in the above test was determined by the method of Junkmann (1954). Progesterone, 17-hydroxyprogesterone caproate and map were used as reference compounds. Immature female rabbits were primed with oestrone as previously described. On the 7th day the compound was given in a single high dose subcutaneously. On the following days 0-025 ^g of oestrone was then given daily subcutaneously. Groups of five animals were killed 3, 7, 10, 13 and 17 days after receiving the test compound. Uterine sections were evaluated as above. Local progestational activity In this test McGinty, Anderson & McCullough's (1939) method was used with progesterone and map as reference compounds. Immature female rabbits were primed as described in the Clauberg test. On the 7th day of the experiment the rabbits were anaesthetized (Nembutal 30 mg/ kg i.v.) and the uteri were exposed. Ligatures were placed at each end of the upper 3 to 4 cm segment of each uterine horn. The upper ligatures were tied tightly and the lower ones left loose without disturbance of the blood supply to the tissue. The test compound (dissolved in 0-1 ml of sesame oil) was intro¬ duced into one of the isolated segments through an incision below the lower ligature. As the contents of the syringe were injected, the lower ligature was tightened. The same volume of sesame oil was injected into the other horn as a control. The rabbits were killed 72 hr later and sections of the isolated segment taken for histological examination. Deciduoma test The method of Elton & Edgren (1958) was used, with progesterone and map as reference compounds. Female hooded rats of the Long-Evans strain (140 to 160 g) were ovariecto- mized, allowed to recover for a week and then primed daily, for 4 days, with 5 µg of oestrone subcutaneously. The test compound was then given subcutane¬ ously daily for the following 9 days. On the 5th day of treatment with the test compound, the left horn of the uterus was exposed and 2-0 mg of histamine Downloaded from Bioscientifica.com at 10/02/2021 12:28:16PM via free access Biological activity of Medrogestone 475 dihydrochloride (in 0-05 ml distilled water) was injected into the lumen, while the right horn served as control. The animals were killed the day after the last subcutaneous injection. The uteri were dissected and the right and left horns weighed separately. Maintenance ofpregnancy test in rats The effect of Medrogestone in maintaining pregnancy was determined according to the method described by Madjerek, de Visser, van der Vies & Overbeek (1960), with progesterone and map as reference compounds. Effect of multiple doses. Groups of six female hooded rats of the Long-Evans strain were placed in cages which contained two mature males per cage. Vaginal smears were taken daily. The day spermatozoa were found in the smears was counted as Day 1 of the pregnancy and the animal was removed from the cage. On the 9th day of pregnancy the rats were ovariectomized and the number of implantations present in the uteri recorded. The test compound was given subcutaneously once daily, or orally twice daily, from the 9th to the 20th day of pregnancy. On the 21st day of pregnancy the uteri were removed through an abdominal incision and the living foetuses counted and weighed. The number of dead foetuses was also recorded. Effect of a single dose. The procedure and reference compounds were the same as when multiple doses were given except that after ovariectomy on the 9th day of pregnancy a single dose of the test compound was administered sub¬ cutaneously in 0-5 ml of sesame oil. The number of live foetuses found in each animal, divided by the number of implantations at the time of ovariectomy, multiplied by 100, was used as an index of the ability of a compound to maintain pregnancy. Ovulation inhibition in rats Ovulation was induced by the method of Wilson & Zarrow (1962). Nore¬ thynodrel, Enovid, Medrogestone and Medrogestone plus Premarin® were tested. Twenty-four-day-old female hooded rats were injected subcutaneously with 30 units of pmsg (pregnant mare serum gonadotrophin). Exactly 56 hr later, they were injected s.c. with 10 units of hcg (human chorionic gonadotrophin). The rats were killed and autopsied 24 hr after the hcg was given. The number of ova expelled from the oviduct was counted under the low power of a light microscope (Rowlands, 1942). The test compounds were given subcutaneously in 0-1 ml of sesame oil with the pmsg, 24 hr before the hcg and with the hcg. The ed50 for ovulation in¬ hibition was the daily dose of the test compound which reduced the number of ova by 50% as compared with the controls. Prevention ofpregnancy test Groups of five adult female hooded rats were injected with Medrogestone for 1 week before they were caged with sexually mature males. Treatment of the females was continued for 2 weeks after which the males were removed from the cages. Vaginal smears were taken during the experiment in order to Downloaded from Bioscientifica.com at 10/02/2021 12:28:16PM via free access 476 C. Revesz and C. I. Chappel detect spermatozoa. The females were checked for pregnancy 1 week after the males were removed. Gonadotrophin suppressant activity This activity was determined in parabiotic rats using the methods of Bunster & Meyer (1933) and Biddulph, Meyer & Grumbeck (1940). Medrogestone was tested alone and in combination with Premarin®. Oestrone was used as the standard. Pairs of 38- to 39-day-old hooded female litter-mates were united by surgical attachment of the lateral skin and abdominal muscles. One of the partners was spayed and given the test compound subcutaneously in sesame oil daily for 10 days. The animals were killed and autopsied on the morning of the 11th day of the experiment. Percentage ovarian inhibition was calculated according to Miyake (1961): 100 [V-C) [V-VJ ' where V average ovarian of the intact partner united with the — weights vehicle-injected spayed female; C = average ovarian weights of the intact partner united with the test compound injected spayed female; and Vt = average weights of the intact partner united with the vehicle- injected intact female. Androgenic and anabolic activity The Herschberger, Shipley & Meyer (1953) assay was used with testosterone propionate, progesterone and map as reference compounds. Twenty-one- to 23-day-old male rats (40 to 45 g) were castrated and given the test compound subcutaneously for 7 days, beginning on the day ofcastration. The animals were killed the day after the last injection and the weight of the levator ani, ventral prostate and seminal vesicles was recorded. Initial and final body weights were also determined. Anti-androgenie effects Some progestational agents have been reported to have strong anti-andro- genic effects. The compounds were assayed for this activity in castrated rats as described by Lerner, Bianchi & Borman (1960).